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Bead-based multiplex serodiagnostics enables simultaneous analysis of antibodies against several antigens. Binding of the antigens onto the surface of the bead, preserving the antigenicity of the antigen is a pivotal step to ensure high sensitivity and selectivity of the assay. Here, a generic method for immobilization of lipopolysaccharide (LPS) antigens from different Gram-negative bacteria to microbeads using non-covalent conjugation has been developed and tested. The method involves coupling of N,N-diethylethylenediamine (DEDA) and derivatives to microbeads. This enhances non-covalent interactions so that LPS is easily immobilized. LPS antigens from the Gram-negative bacteria Actinobacillus pleuropneumoniae (APP) and Salmonella enterica serogroup B (Sal. B) were immobilized on the DEDA-coupled microbeads. In parallel, the same LPS antigens were coupled to beads using two previously reported methods. The performance of microbeads coupled with antigen using the different methods was compared by measuring antibodies in positive and negative serum samples from pigs. DEDA-beads coupled with LPS detected pathogen specific serum antibodies with equal or higher sensitivity and specificity compared to the other coupling methods used in this study. Furthermore, derivatives of DEDA, where the tertiary amine was alkylated with a methyl (m-DEDA) and ethyl group (e-DEDA) to give a positively charged tetraalkylammonium group, were compared with DEDA for the binding of LPS antigens. Here, it was concluded that the DEDA-modified bead was most efficient in the binding of LPS antigens from two Actinobacillus pleuropneumoniae serovars and Salmonella enterica serogroup B.

We developed and made a preliminary validation of a bead-based multiplexed immunoassay for simultaneous detection of porcine serum antibodies to Actinobacillus pleuropneumoniae serovars 1, 2, 6, 7, and 12. Magnetic fluorescent beads were coupled with A. pleuropneumoniae antigens and tested with a panel of serum samples from experimentally infected pigs and with serum samples from uninfected and naturally infected pigs. The multiplex assay was compared to in-house ELISAs and complement fixation (CF) tests, which have been used for decades as tools for herd classification in the Danish Specific Pathogen Free system. Assay specificities and sensitivities as well as the corresponding cutoff values were determined using receiver operating characteristic (ROC) curve analysis, and the A. pleuropneumoniae multiplex assay showed good correlation with the in-house ELISAs and CF tests with areas under ROC curves ≥ 0.988. Benefits of multiplexed assays compared to ELISAs and CF tests include reduced serum sample volumes needed for analysis, less labor, and shorter assay time.